1. Field of the Invention
The present invention relates to the diagnosis of disorders or dysfunctions characterized by autoimmune responses to the enzyme class of transglutaminases. The present invention provides a novel open structure of the transglutaminases in a stabilized form which renders new epitopes accessible for antibody-binding.
2. Description of the Relevant Art
Transglutaminases (TG) are enzymes that exist in higher vertebrates including the human body as a family of nine members, including the inactive structural protein band 4.2. They fulfil a large variety of physiological functions e.g. in coagulation, inflammation, cell differentiation, apoptosis, cell-cell and cell-matrix interactions, and wound healing. In the presence of Ca2+ transglutaminases may develop their catalytic activity, the formation of inter- and intra-molecular γ-glutamyl-ε-lysine bonds, also called iso-peptide bonds, between the side chains of peptidic glutamine and lysine. In general the results of this activity are high-molecular weight protein aggregates. Transglutaminases 1, 3 and 5 are mainly involved in skin-formation, factor XIII (plasma-transglutaminase) is active in blood coagulation and wound healing, transglutaminase 6 has mainly been found in neuronal tissues and transglutaminase 4 in semen fluid. Transglutaminase 2 (tissue transglutaminase, TG2) is ubiquitously present in various tissues. Besides its cross-linking activity TG2 may also deamidate the carboxyamide group of peptidic glutamine, especially under slightly acidic conditions as well as in the absence of lysine or other primary amines. As the deamidation product is a carboxyl group, a negative charge is introduced into the peptide which results in a significant change of peptide properties. Further TG2 has GTP-hydrolysing activity and functions as a G-Protein. Even kinase activity is described. TG2 plays a role in several diseases like celiac disease, fibrosis, cancer, and neurological disorders.
In celiac disease, a chronic inflammation of the small intestine and an autoimmune disease, TG2 plays a multifunctional role. Digestion of proteins present in our food is performed by proteolytical cleavage to amino acids and small peptides, which can be resorbed by the intestinal mucosa. Cereal proteins like gluten show a high content of the amino acid proline. But the enzymes responsible for protein degradation in the digestive tract are not able to hydrolyse proline-rich proteins into amino acids and small peptides. Consequently rather long peptides, like gliadin peptides are present in the mucosa. Due to their size they cannot be resorbed, but they are still able to pass the epithelium and to reach the so called lamina propria, a connecting tissue between epithelium and muscular tissue. In the lamina propria extracellular TG2 is present. Now an essential step in celiac disease pathophysiology takes place: TG2 deamidates gliadin peptides. In their deamidated form gliadin peptides are recognized by the HLA-DQ2 and HLA-DQ8 receptors of immune cells. This recognition leads to the triggering of the inflammatory response which in the final stage of the disease manifests in a complete villous atrophy of the intestinal mucosa. TG2 not only catalyses gliadin deamidation, it serves also as autoantigen in celiac disease. Patients who suffer from celiac disease develop autoantibodies against TG2. If patients successfully follow a strict gluten free diet anti-TG2-autoantibody titers decrease. Therefore TG2 is not only used as antigen for autoantibody-detection in the diagnosis of celiac disease, but also in the follow up and control of the gluten free diet. TG2-autoantibodies are also found in other diseases like diabetes type 1 or psoriasis and may therefore be used in non-celiac disease diagnostics too.
Interestingly in other indications developing along or in consequence to celiac disease further autoantibodies against transglutaminases can be found: Dermatitis herpetiformis, a gluten sensitive skin disorder is characterized by the presence of autoantibodies against epidermal transglutaminase (TG3) whereas in glutensensitive ataxia autoantibodies against neuronal transglutaminase (TG6) have been found.
Objective of the present invention is to provide a more sensitive method for detecting autoimmune diseases characterized by the presence of transglutaminase specific autoantibodies.
The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures and the examples of the present application.